Instrumental techniques for identifying one or more components of a complex mixture are of use in diverse fields. Mass spectrometry is a robust and versatile instrumental technique that provides the ability to rapidly sort through complex mixtures and identify the components of the mixture.
The use of mass spectrometry to analyze mixtures of proteins, peptides, oligonucleotides, and noncovalent complexes is rapidly being adopted in biological research, especially for proteome characterization, protein profiling and genomics. There is a well-recognized need for the high throughput identification of these and other species, for example proteins and their post-translational modifications that are, for example, up-regulated or down-regulated in response to a specific external stimulus, the onset of disease, or normal aging.
The conventional approach to analyzing complex biological mixtures involves the high resolution separation of components of the mixture using 2D polyacrylamide gel electrophoresis followed by their one-at-a-time excision and characterization, increasingly exploiting mass spectrometry. Additional information is generally gathered in the form of a correlation between the peptide masses for peptide fingerprinting (e.g., their common origin from a single protein), or by partial peptide sequencing. However, even with complete automation of separations and sample processing there are practical limitations upon the throughput of these methods.
Mass spectrometry is also of interest for the identification of microorganisms. Chemotaxonomy of microorganisms based upon their spectroscopic, spectrometric, and chromatographic characteristics represents a useful method for the identification of microorganisms such as yeasts, fungi, protozoa, viruses and bacteria. Typically, such chemotaxonomic methods are based upon instrumental methods that provide “fingerprint” spectra or chromatograms (i.e., spectra or chromatograms that are unique to each type of microorganism). Such fingerprinting methods include mass spectrometric methods, infrared spectroscopy, ion mobility spectrometry, gas chromatography, liquid chromatography, nuclear magnetic resonance, and various hyphenated techniques such as gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-Fourier transform infrared spectroscopy (HPLC-FTIR).
Recently, mass spectrometric techniques have been developed for generating specific protein profiles for various biological agents. These techniques generally employ electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) of protein extracts followed by mass spectrometric (MS) or tandem mass spectrometric (MS/MS) analysis. ESI and MALDI are ionization techniques that have enabled dramatic progress to be made in performing mass spectrometry on large biomolecules including proteins. MALDI, combined with time-of-flight mass spectrometry (TOF-MS), has been used to differentiate biological agents using a crude protein extract. For example, Krishnamurthy and coworkers have developed methods and apparatus for identifying biological agents through the automated detection of biomarkers such as proteins released from extracts or whole intact biological agents that may be present in an environmental or biological sample. See, U.S. Pat. No. 6,558,946.
Instrumental fingerprinting methods, such as mass spectrometry, tend to suffer from irreproducibility due to both instrumental and environmental factors. For example, continued use of a mass spectrometer leads to contamination of the ion optics and thus can lead to alterations in the appearance of a microorganism's fingerprint mass spectrum. Changes in microorganism characteristics due to environmental factors, such as the patient from which the organism is isolated, or the growth medium used to culture the microorganism, can also alter the appearance of a microorganism's fingerprint spectrum. Irreproducibility of spectral data due to instrumental and environmental sources makes it difficult to classify or identify microorganisms based on fingerprint spectral patterns.
An effective method for characterizing the components of a complex mixture must be rapid, sensitive, selective, and cost-effective. The use of higher mass accuracy mass measurements has the potential to greatly speed characterization of the components of complex mixtures. Sufficiently high mass measurement accuracy, in principal, can enable the identification of a protein from a single peptide mass. Moreover, the methods should be reliably repeatable across an array of similar samples that are analyzed at different times. To this end, methods have been developed for calibrating analytical instruments. For example, U.S. Pat. No. 5,710,713 describes a method for determining whether, and by how much the sensitivity or bias of an mass spectrometer may have drifted outside an acceptable, application-defined tolerance level throughout a spectral region of interest. Generally, this method involves determining relative instrument bias by using spectra, of at least a single standard, acquired at different times, or on different instruments. The observed changes in the spectra are used to generate a mathematical function of the change in instrument bias.
In another approach described in U.S. Pat. No. 6,498,340, a mass spectrometer is calibrated by shifting the parameters used by the spectrometer to assign masses to the spectra in a manner which reconciles the signal of ions within the spectra having equal mass but differing charge states, or by reconciling ions having known differences in mass to relative values consistent with those known differences. The method makes use of data along the X-axis (m/z) only and does not utilize the Y-axis data (intensity). Moreover, the method does not identify the components of complex mixtures by comparing the processed mass spectra to a library reference set of similarly processed mass spectra.
Processing of more complex mixtures for ever higher throughput analyses, such as the analysis of complex mixtures of biological agents, e.g., microorganisms, results in much greater demands on mass spectrometry, in terms of speed, resolution, mass measurement accuracy, and data-dependent acquisition. Moreover, there is need for a method that can be practiced by a technician in the field, hospital, or clinical laboratory or in bioprocessing and manufacturing. As such, calibration schemes that can enable higher mass accuracy measurements to be accomplished over a wide range of conditions play an essential role in the successful application of mass spectrometry to protein identification from complex peptide mixtures. The present invention meets these needs.